A neurotoxic syndrome called Intermediate
A neurotoxic syndrome called “Intermediate syndrome” has been described after acute cholinergic crisis, which has been interpreted as the result of pre- and postsynaptic disruptions of neuromuscular transmission (Senanayake and Karalliedde, 1987). Other toxic effects with unknown molecular targets have been described but poorly defined (COT, 1999; Jamal et al., 2002).
Several enzymatic components of phenyl PVase activity have been discriminated using irreversible inhibitors: mipafox (OPIDN-inducer), paraoxon (non OPIDN-inducer) and phenylmethylsulfonyl fluoride (PMSF) (Mangas et al., 2011, 2012, 2012b, 2014) in membrane and soluble fractions. PMSF is an NTE inhibitor that protects against OPIDN development when dosed before a neuropathic dose of a neuropathic OP, but PMSF enhances neuropathy severity when dosed after a low non-neuropathic dose of a neuropathy inducer (Lotti et al., 1991; Pope and Padilla, 1990).
In the membrane fraction, four enzymatic components (EPα, EPβ, EPγ and EPδ) were discriminated using the combined information of the inhibitory kinetic properties with several irreversible inhibitors (Mangas et al., 2014). The characteristics of these fractions are summarized in Table 1. EPα is highly sensitive to mipafox and paraoxon, but is resistant to PMSF, and is spontaneously reactivated when inhibited with paraoxon. EPβ is sensitive to paraoxon and PMSF, but is resistant to mipafox. EPγ, is resistant to paraoxon, sensitive to mipafox and PMSF, and matches the operational criteria of being “NTE” (Johnson, 1982). EPδ is resistant to all the assayed inhibitors and it is related to the IRE (inhibitor resistant esterase) as described by Johnson and Richardson (1983). The strategies adopted to discriminate these components in this work are shown in Table 2.
Moreover, the soluble fraction of chicken TC 14012 has been studied by Benabent and coworkers (2014), who showed that phenyl valerate may interact with cholinesterase activity, and several PVase components were discriminated (Eα, Eβ, Eγ). The interactions of Eα, Eβ and Eγ with PMSF (Mangas et al., 2012) suggest that they could play a role in the potentiation/promotion phenomena described by Pope and coworkers (Pope and Padilla, 1990) and by Moretto and Lotti laboratory (Lotti et al., 1991). Component Eα (PVase activity resistant to PMSF) is inhibited by acetylthiocholine, ethopropazine and iso-OMPA. This was considered to indicate that both substrates may interact on the same protein, and that all or part of Eα PVase activity might be due to butyrylcholinesterase (BuChE). However, component Eβ (resistant to mipafox) showed no interaction with acetylthiocholine. In the above work, component Eγ (resistant to paraoxon) was not studied because no cholinesterase activity was observed at the paraoxon concentration used to discriminate component Eγ. An enriched fraction with component Eα was prepared by different separation methods of the native protein (Mangas et al., 2014b), and was analyzed by LC–MS/MS and bioinformatic analyses (Mangas et al., 2017). It showed that BuChE was the only candidate responsible for virtually all Eα PVase activity. As a result of these findings, we studied the human BuChE and showed that it has PVase activity and did some characterization about their kinetic behavior (Mangas et al., 2017b). Kohli et al (2007) had observed that a biosensor containing preparation of commercial BChE and tyrosinase showed electrochemical response with phenyl valerate. This might be interpreted by a catalytic activity of BuChE with phenyl valerate. However the specific source of the enzymes in that work were commercial and no detail about it specie origin and purity were reported, and therefore, other proteins and factors may be influencing. With our current knowledge, we can interpret that those observations could be due to hydrolysis caused by BChE but other factors cannot be discarded.
Materials and methods